Mitigation of intrahepatic cholestasis induced by 17α-ethinylestradiol via nanoformulation of Silybum marianum L.

Background Cholestasis is an important predisposing factor for hepatocyte damage, liver fibrosis, primary biliary cirrhosis, and even liver failure. Silybum marianum L. (SM) plant is used in teas or eaten in some countries due to its antioxidant and hepatoprotective properties. Because of its low and poor oral bioavailability, so we improve the therapeutic activity of Silybum marianum L. extract (SM) by studying the potential effects of nanoformulation of Silybum marianium L. extract (nano-SM) on 17α-ethinylestradiol (EE)-induced intrahepatic cholestasis. Methods Thirty female Sprague-Dawley rats were divided into 5 groups (6 rats/group). Group I: Rats were received the treatment vehicle and served as normal group. Group II:Rats were injected daily with EE (10 mg/kg) for five successive days. Group III-V: Rats were injected daily with EE (10 mg/kg) and treated with either Ursodeoxycholic acid (UDCA) (40 mg/kg), SM (100 mg/kg) and nano-SM (100 mg/kg) orally once/day throughout the trialfor five successive days, respectively. Results Nano-SM greatly dampened the increase in serum levels of total and direct bilirubin, alanine aminotransaminase, aspartate aminotransaminase, and alkaline phosphatase caused by EE. Furthermore, nano-SM increased the hepatic contents of reduced glutathione (GSH) and catalase (CAT) and also upregulated the relative hepatic gene expressions of Rho-kinase (ROCK-1), myosin light chain kinase (MLCK), and myosin phosphatase target subunit (MYPT1) compared to the EE-induced group. Administration of nano-SM reduced hepatic lipid peroxidation and downregulated the relative hepatic expressions of the nuclear factor-kappa B (NF-ҡB) and interleukin-1β (IL-1β). In addition, nano-SM improved the histopathological changes induced by EE. Conclusion Nano-SM possessed a superior effect over SM, which can be considered an effective protective modality against EE-induced cholestatic liver injury through its antioxidant, anti-inflammatory activities, and enhancing bile acid (BA) efflux.

Mitigation of intrahepatic cholestasis induced by 17α-ethinylestradiol via nanoformulation of Silybum marianum L.

Background
Cholestasis is a clinically pathogenic condition in which toxic bile acid (BA) accumulates excessively in the liver [1], autoimmune liver diseases [2], liver cell necrosis, liver fibrosis, primary biliary cirrhosis [3], and even liver failure [4].Patients with chronic liver disease had a 10.26% overall cholestasis incidence rate [5], and the death rate for cholestasis patients is 7.8% [6].
Synthetic estrogen 17-ethinylestradiol (EE) is often employed to create an experimental model of intrahepatic cholestasis to investigate the underlying molecular mechanism of estrogen-induced cholestasis (EIC).When EE activates the estrogen receptor α, BA transporters are suppressed, and BA synthesis enzymes are altered, resulting in cholestatic liver damage [7,8].BA transporter malfunction, or oxidative stress, which may cause inflammation, can be the key contributors to the etiology of intrahepatic cholestasis [9,10].
Currently, limited effective drugs are accessible for cholestasis therapy.Ursodeoxycholic acid (UDCA) is a safe and effective treatment for cholestasis and can effectively treat 50% of primary biliary cholangitis patients; while 40% of patients showed poor responses, and 5-10% of them suffered from intolerance.Nevertheless, some patients suffering from primary biliary cholangitis still show a progressive disease that requires liver transplantation or, in some cases, can lead to death, despite UDCA treatment [11].Therefore, searching for a new drug avenue for treating or protecting against cholestasis is in great demand.
Natural products have recently been considered a crucial aid in the search for innovative therapeutic drugs to treat and prevent cholestatic liver disorders [12,13].Milk thistle, or Silybum marianum L. (SM), is a member of the Asteraceae family with purple blooms, light green leaves, and thorny, mallow-like stems [14,15].The liver cells regenerate more quickly due to its involvement in expelling poisons that bind to the organ [16].SM is used as a tea or eaten in several countries in the Middle East and North Africa due to its ability to support patients with liver cirrhosis, alcoholic hepatitis, alcoholic fatty liver, liver toxicity, and viral hepatitis [17][18][19].
Despite the therapeutic benefits of silymarin, its weak biochemical properties (a lack of solubility in water) and limited bioavailability caused unsatisfactory and inconsistent therapeutic results, which leads to more instances of drug-drug interactions when other concurrent medications are given [27,28] which led to search for innovative method to solve such problems as using nanotechnology.Nanotechnology offers the chance to complement natural remedies by facilitating the identification, manufacture, and implementation of a wide range of therapeutic strategies to improve health and lessen the severity of various diseases [25].Accordingly, this study aimed to improve the therapeutic activity of SM by studying the potential protective effects of nano-SM on EE-induced intrahepatic cholestasis in Sprague-Dawley rats.

Preparation of the plant extract
SM seeds were collected at the end of February 2021 from NRC Farm, Cairo, Egypt and identified by Dr. Ahmed Ali Muhammed, Department of Horticultural Crops Technology, National Research Centre (NRC).Herbal specimen was kept at NRC herbarium (voucher specimen #6411).Relevant permissions were obtained for the collection of SM seeds in accordance with relevant institutional, national, and international guidelines and legislation.
The ethanolic extract was carried out, as stated by [29], with various modifications.In brief, 200 g of dried SM seeds were combined with 1 L ml of ethanol (70%) and stirred overnight at room temperature.Then, the ultrasonic probe (160 W power, 20 kHz frequency, 50% pulse; Sonics, Vibra, Cell, USA) was utilized for 15 min.The extract was then split apart by centrifugation at 3000 rpm for 15 min, followed by evaporation of the solvent at 50 °C using a rotary evaporator (Büchi R20, Switzerland).Finally, the extract was collected and kept in a volumetric flask at 20 °C until it was needed for the experiment, and the yield % was recorded.

Chemical analysis of SM
Silymarin content was detected, as stated by [30].The total carbohydrate content of plant seeds was evaluated using the phosphomolybdic acid technique, as stated by [31].The total phenolic content of SM was assessed using the spectrophotometric technique of [32,33].Tannin contents were evaluated using the Folin-Ciocalteu reagent technique, as stated by [34].Crude protein content was estimated by multiplying seed nitrogen content by 6.25.At the time of harvest, dry seed mineral content was assessed to determine the total nitrogen (N) using the micro-Kjeldahl technique [35].Phosphorus (P) and Potassium (K) were determined and estimated as percentages of dry weight as stated by [36].The contents of Fe, Mn, and Zn were evaluated using atomic absorption spectrophotometer and were calculated in ppm [37].established a technique for determining the quantity of fatty acids in seeds.Amino acids were extracted and quantified using Amino Acid Analyzer (AAA 400 INGOs Ltd) according to the methods evaluated by [38,39].Total flavonoids in crude extract were determined using the [40] method, modified by [41].Ascorbic acid (Vitamin C) was determined using the spectrophotometric method of [42].

Antioxidant assay (DPPH radical scavenging assay)
Using the DPPH radical scavenging test, the antioxidant activity of SM was determined as follows: The antioxidant scavenging activity of SM was investigated using the 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH), which was created by mixing 1.5 mL of different dilutions of SM with 1.5 mL of a 0.2 mM methanolic DPPH solution [43].After 30 min of incubation at 25 °C, recorded as A (sample) was measured using a spectrophotometer at 520 nm.In the absence of SM, a blank experiment was conducted using the same methodology.Absorbance was recorded as A (blank).The % inhibition of both solutions' free radical-scavenging activity was estimated using the equation below: % inhibition = 100 (A (blank)-A (sample)) / A (blank).The IC50, or the concentration of SM necessary to elicit a 50% reduction in initial DPPH concentration, was used to measure antioxidant activity.As a control, ascorbic acid was used.Each value was measured three times for accuracy.

Preparation of Silybum marianium L. Nanoformulation
The nano-SM was prepared using the solvent Emulsification-Diffusion process with novel modifications, as stated by [44].The Egyptian Patent Office of the Academy of Scientific Research and Technology has a running patent request on this novel method with number 1956/2020, which contains the details of the nano preparation process and the associated measurement data.

Nanoparticles measurement techniques Transmission electron microscopy (TEM)
The samples were investigated using a transmission electron microscope (TEM) (JEM − 1234) with a 120 KV operating voltage, a magnification power of 600,000 x, a resolving power of 0.3 nm, a CCD camera, and a programmed heating/cooling facility ranging from − 190 0 C to 1000 0 C. On a copper grid with a carbon coating, samples were kept [45,46].

Zeta sizer nano ZS
Using a Zeta Sizer Nano ZS (Malvern Instruments Inc., Southborough, MA) at 25 °C, the Z-average hydrodynamic diameter of the samples under investigation was evaluated.The size distribution (by number), polydispersity index (PdI), and ζ-potential of nano-SM were investigated.

Animals
Female adult Sprague-Dawley rats (100-140 g), aged 8 to 11 weeks, were purchased from the Schistosome Biology Supply Center, Theodor Bilharz Research Institute, Giza, Egypt.Rats were housed under 12 h light & dark cycles with free access to water and food at a temperature of 25 ± 2 °C and humidity of 50 ± 15%.

Drugs and doses
EE (Folone (R); Misr Company for Pharmaceuticals, El-Asher Men Ramadan, Cairo, Egypt, batch number: 19,606,007), at a dose of 10 mg/kg, was administrated subcutaneously once/day for five days [47].EE is used to establish the intrahepatic cholestasis model in adult female Sprague Dawley rats according to [48][49][50].

Diet composition
The balanced diet, salt mixture and vitamin mixture were prepared according to [51] as described below (Table 1):

Experimental animal design
A total number of 30 female Sprague-Dawley rats were divided into 5 groups (6 rats / group) as described below: (I) Normal group.(II) EE-induced intrahepatic cholestasis group.(III) EE + UDCA-treated group.(IV) EE + SM-treated group (SM was administered at a dose of 100 mg/kg) [44].(V) EE + nano-SM-treated group (nano-SM was administered at a dose of 100 mg/kg) [44].Twenty-four hours after the administration of treatments, rats will be sacrificed by decapitation under sodium pentobarbital (50 mg/kg, i.p.) anesthesia.Sera were collected for liver function assays.The livers were immediately removed for oxidative stress markers assessment and RNA extraction.Part of the livers was fixed in 10% formalin for histopathological and immunohistological studies.

Oxidative stress markers assessment
For oxidative stress markers assessment, liver tissue was homogenized, followed by centrifugation for 10 min at 600 g and then for 20 min at 10,000 g at 4 o C. The supernatants were tested for reduced glutathione (GSH), catalase (CAT), and lipid peroxidation, which was expressed by malondialdehyde (MDA) formation, using commercial kits (Biodiagnostics, Egypt).

Histopathological examination
10% formalin-fixed liver samples were processed and stained with hematoxylin-eosin (H&E) stain.The histopathological changes were examined under a light microscope (magnification x400).

Real time PCR for BA efflux and inflammatory markers
RNA was extracted from homogenized liver tissues and reverse transcripted to cDNA using QIAamp-RNA-Mini-Kit (Qiagen, Germany) and QuantiTect-Reverse-Transcription-kit (Qiagen, Germany), respectively.Finally, the relative expressions of ROCK, MLCK, MYPT1, NF-ҡB, and IL-1β genes were done using QuantiTect-SYBR-Green-PCR-kit (Qiagen, USA).β-actin was used as a reference gene.Primers were designed by Thermo (Table 2).The expressions of ROCK, MLCK, and MYPT1 are used to assess the ROCK/MLCK/myosin pathway, which is responsible for bile canaliculi (BC) spontaneous contractions and dilations, which are essential for bile acid (BA) efflux [52].While the expressions of NF-ҡB, and IL-1β are used to assess the inflammatory process, which is one of the key contributors to the etiology of intrahepatic cholestasis [9,10].

Immunohistochemical evaluations of IL-6
Immunohistochemical evaluations of IL-6 were performed using an anti-IL-6 antibody (Santa Cruz Biotechnology, USA) and DAKO-EnVision-FLEX detection kit [53].The percentages of positive cytoplasmic staining for IL-6 in 10 fields (magnification x400) were calculated for each rat.

Statistical analysis
SPSS, software package version 16.0 (Chicago, USA), One-way-ANOVA-test followed by Tukey-hoc-test was used to determine the significant difference between the different groups.Results were expressed as mean ± SE and considered significant when the P < 0.05.

Results and discussion
The results of this experiment revealed that nano-SM could improve the therapeutic activity of SM and can be used as an effective modality in protecting against EEinduced-cholestatic liver injury, as the limitations of the SM were resolved by using thisnano-formulation.

Chemical analysis of SM
SM had an extraction yield of 50 g/200 g dried plant (Table 3), which was corroborated by [54,55].Silymarin concentration in SM was 51.43 mg/g.This finding has been supported by other researchers [56][57][58].The  total carbohydrates were 43.79%, and the crudeprotein was 24.36%, with total phenolic, tannin, ascorbic acid, and total flavonoids averaging 31.51mg/g, 3.41 mg/g, 2.67 mg/g, and 19.95 mg/g, respectively.SM has a protein content of 25-30% [59][60][61][62].Protein and total carbohydrates are abundant in the seeds.Antioxidant qualities are attributed to flavonoids and phenolic acids due to the presence of hydroxyl groups in their structures; these compounds play a crucial role in the body's defense mechanism versus oxidative damage produced by endogenous free radicals [63].Vitamin C (ascorbic acid) is engaged in a variety of metabolic processes and is required for collagen formation; it also improves blood circulation and acts as an antioxidant [64] (Table 3).Micronutrients, also known as trace elements, are a class of nutrients found in small amounts in the diet.Because of growing evidence of marginal or insufficient intakes among the population, the nutrition community is particularly interested in the trace element composition of diets [65].According to Table 4, K, N, and P made up 5.2%, 3.94%, and 0.89% of the total SM seed, respectively.Fe, Zn, and Mn concentrations were found to be 0.91, 0.089, and 0.088 ppm, respectively.The results agreed with [66,67], who found that SM seeds contain high mineral content of K, N, P, Fe, Zn, and Mn elements, which are necessary for proper human nutrition.
In Table 4, the primary oils found in SM were linoleic acid, oleic acid, palmitic acid, and stearic acid, according to GC analysis.Linoleic acid, oleic acid, palmitic acid, and stearic acid made up 48.2%, 29.40%, 12.90%, and 11.80% of the total SM seed, respectively.Seed oil is the plant's second most significant product, and its fatty acid content is comparable to that of seed oil (linoleic acid > oleic acid > palmitic acid > stearic acid) [68].
The nutritional value of food, particularly protein, is determined not just by its amino acid profile but also by the amounts of essential amino acids present [69].The amino acid content of SM is shown in Table 4, and it is divided into five categories.SM is a rich source of phenylalanine (5.79 g -1 /kg).On the other hand, it is a poor source of tryptophan (0.99 g -1 /kg).SM was also shown to have significant levels of lysine, valine, and histidine with 4.98, 3.97, and 1.89 g -1 /kg, respectively.The previous investigation concurred with these findings [67].

Antioxidassayssay (DPPH radical scavenging assay)
The antioxidant activity of SM in Fig. 1 was investigated using the DPPH assay, which showed that the ascorbic acid was 99.7 ± 0.32 mg/L and the ethanolic extract had lower IC 50 than ascorbic acid, which was 89.1 ± 0.45 mg/L.Due to their potent DPPH radical inhibition, the DPPH radical scavenging capability of SM was shown to have fairly excellent free radical scavenging capacities [70].

Nanoparticles measurement techniques Transmission Electron Microscopy (TEM) analysis
Transmission electron microscopy (TEM) has been used as a based technique for investigating the morphology of SM, which revealed spherical vesicles with a diameter of around 50 nm (Fig. 2).

Refractindexndex Zeta Sizer
Nanomaterials differ from their bigger counterparts regarding their physical, chemical, and biological characteristics.Early detection and prevention, better diagnosis,   74].Furthermore, these nanoparticles reduce toxicity, retain therapeutic properties, reduce side effects, demonstrate EPR (enhanced permeability and retention) impact, and reduce medication formulation dosage.These factors combine to overcome the limitations of SM, improve its therapeutic activity and make nano-SM an effective modality in protecting against EE-induced-cholestatic liver injury.

Liver injury Assessment
Cholestasis is a multi-factorial complex disease with various manifestations.Intrahepatic cholestasis is a common symptom of drug-induced liver injury [75].Estrogen and its metabolites (17β-estradiol) may cause intrahepatic cholestasis in pregnant and pre-menopausal females taking oral contraceptives or hormonal replacement therapy [76].SM possesses hepatoprotective properties due to its anti-inflammatory, antioxidant, and membranestabilizing effects.Moreover, it was also shown to guard against estrogen or taurolithocholate-induced cholestasis through its anti-cholestatic effect, including the bile salt pool size and HCO 3 -output normalization and bile salt transport regulation [77,78].
Table 5 illustrated that the EE-induction of intrahepatic cholestasis led to a noticeable significantly increase (p < 0.05)in serum ALT, AST, ALP, and total and direct bilirubin levels by nearly 2.35, 1.80, 2.21, 7.98, 9.88, and 7.03-folds respectively compared to the normal group.This is in agreement with previous studies [79,80], indicating loss of hepatic cell membrane integrity and leakage of bilirubin and liver enzymes into the blood circulation.Notably, treatment with UDCA significantly suppressed (p < 0.05) the EE-induced elevation in serum ALT level by 39.77% compared to the EE-induced intrahepatic cholestasis group.On the other hand, treatment with UDCA normalized serum AST, ALP, and total and direct bilirubin levels.Administration of SM significantly reduced the elevation (p < 0.05) in serum ALT by 34.85%, AST by 22.78%, ALP by 29.86%, total bilirubin by 51.35%, and direct bilirubin by 27.38%, compared with the EEinduced intrahepatic cholestasis group.However, treatment with nano-SM resulted in the normalization of liver function markers and bilirubin contents.The reduction of liver enzymes and bilirubin by nano-SM may be due to its membrane-stabilizing activity [77,78].

Oxidative stress markers assessment
Oxidative stress and inflammation are crucial in the early induction of liver injury associated with cholestasis [81], where BA accumulation induces liver injury followed by neutrophil recruitment and infiltration [82].
EE-induced cholestasis resulted in a significant depletion in hepatic GSH and CAT by 2.42 and 1.96-folds, respectively, along with a significant increase (p < 0.05) in lipid peroxidation, which is expressed as hepatic MDA by 2.79-fold.These results were consistent with the earlier studies of [83,84].Nevertheless, treatment with UDCA normalized hepatic GSH and MDA and significantly elevated (p < 0.05) hepatic CAT by 53.56%, compared with the EE-induced intrahepatic cholestasis group.Moreover, treatment with SM resulted in a significant increase (p < 0.05) in hepatic GSH and CAT by 56.46% and 21.41%, respectively, with a significant reduction (p < 0.05) in hepatic lipid peroxidation by 36.64%, compared with the EE-induced intrahepatic cholestasis group.In addition, nano-SM administration normalized all hepatic oxidative stress markers (Fig. 4), indicating the potent antioxidant activity of the SM and nano-SM.This agreed with previous studies [85,86] which revealed that SM had antioxidant and hepatoprotective properties.

Real time PCR for BA efflux and inflammatory marker
Biliary secretion is a multi-step process that includes the translocation of BA across the cell membrane, the cytoplasm, and the canalicular membrane to the canalicular lumen [75].Because bile canaliculi contraction is essential for BA efflux, Drugs that would improve contractile movement may be a promising candidate for intrahepatic cholestasis treatment.
Compared to the normal group, EE deteriorated the BA reflux as manifested bya significant reduction (p < 0.05) in ROCK-1, MLCK, and MYPT1 by 2.56, 2.50, and 1.39folds, respectively (Fig. 5), reflecting bile canaliculidilation and BA accumulation.However, treatment with SM significantly increased (p < 0.05) ROCK-1 by 74.36%, MLCK by 75% and MYPT1 by 83.33%, compared with the EE-induced intrahepatic cholestasis group (Fig. 5).On the other hand, administration of UDCA or nano-SM normalized ROCK-1, MLCK, and MYPT1indicating the restoration of normal bile canaliculi contraction leading to normal BA flow.These findings concur with [52], who stated that BA flow depends on bile canaliculi's spontaneous and rhythmic contractions.
Inflammation occurs in EE-induced intrahepatic cholestasis, and the increased inflammatory cytokines accelerate cholestasis development and progression [87].NF-κB is a crucial transcription factor that regulates several inflammatory cytokines, including IL-6 and IL-1β [88].

Histopathological examination
The liver section of the normal group revealed normal liver architecture (Fig. 7A).However, EE administration led to severe dilatation and congestion in central and portal veins, severe degeneration in the surrounding hepatocytes, and diffused Kupffer cell proliferation with sinusoidal inflammatory cell infiltrations surrounding the obliterated bile ducts (Fig. 7B).UDCA-treated rats showed mild congestion in the portal vein, mild degeneration in the surrounding hepatocytes, mild Kupffer cell proliferation, and moderate newly formed bile ductules in the portal area (Fig. 7C).Treatment with SM showed moderate congestion in the portal vein, moderate degeneration in the surrounding hepatocytes, moderate Kupffer cell proliferation, and moderate newly formed bile ductules in the portal area (Fig. 7D).Administration of nano-SM revealed almost normal hepatocytes with mild congestion in the central vein and mild inflammatory cell infiltration (Fig. 7E).Our results align with a previous study [91], which demonstrated that SM nanoformulation could improve liver histopathology regarding connective tissue deposition and cellular infiltrates.

Conclusion
The current study has shown that Nanoformulation could improve the therapeutic activity of silymarin and can be considered a promising and effective treatment in protecting against EE-induced cholestatic liver injury, with significant potential for future advancements through its antioxidant and anti-inflammatory activities and enhancing BA efflux.Consequently, Nano-SM may be a potentially beneficial drug for intrahepatic cholestasis prevention.In addition, we also have to take into the consideration the oral bioavailability of Nano-SM in human and the application of drug delivery system for the successful outcomes in clinical trials.

Table 1
Ingredient composition of the experimental diet *gm/kg diet

Table 2
The primer sequences for real-time PCR

Table 3
Yield and chemical composition of Silybum marianum L. seeds

Table 4
Minerals content, Fatty acids composition (%), and Amino acids composition of Silybum marianum (L.) seeds when converted SM was converted to nanoparticles, the produced nanoparticles with Z-average (d.nm) = 45.27,Pdl= 0.23, and particlesize (d.nm) = 22.67 with % Number = 100% and ζ-potential= -40.40 which exhibited a unique attribute of high surface area to volume ratio according to size distribution, polydispersity index, and tive or positive), they repel each other, lowering the likelihood of aggregation.Physically constant particles have a ζ-potential larger than 30 mV (positive or negative), but a ζ-potential close to 20 mV (positive or negative) suggests low colloidal suspension stability, and values in the range of 5 mV to + 5 mV indicate fast particle aggregation [73,Table 5 Effect of SM or Nano SM on serum biochemical makers